The brain is a highly autoregulated organ in which cerebral blood flow remains relatively constant over a wide range of arterial pressures. Isolated, cannulated cerebral arteries subjected to increasing levels of transmural pressure develop active tone, and constrict as pressure is elevated. Our studies suggest that the vascular endothelium senses pressure increases releasing a transferable vasoactive substance which depolarizes the vascular muscle cell. We will use a bioassay preparation to assess the mechanisms of action of endothelial factors on the electrical and mechanical properties of isolated arterial segments. Isolated cerebral arterial muscle cells will be superfused with the effluent from pressurized cerebral arterial to examine the effect of endothelial factors on whole cell K+ and Ca2+ current measured via patch clamp techniques. Microsomal fractions of arteries loaded with radiolabeled arachidonic acid produce a HPLC fraction which contracts isolated arteries. Production of this fraction and pressure induced activation of cerebral arteries are both blocked by selective inhibition of cytochrome P-450 arachidonic acid metabolism. We will assess the effect of inhibitors of arachidonic acid metabolism with (specific reference to the cytochrome P-450 pathway) on cerebral blood flow measured by laser-Doppler flowmetry through a pial window, and electrical and mechanical responses to pressure in isolated cerebral arteries. We will also assess the effect of this vasoactive HPLC fraction on K+ and Ca2+ currents in isolated muscle cells. To explore the mechanisms by which local metabolic factors override pressure induced activation, the influence of oxygen and oxygen free radicals on electrical and mechanical coupling and endothelial muscle interactions in isolated, pressurized cerebral arteries will be studied. We will also explore the direct effects of reducing PO2 on K+ currents in isolated cerebral arterial muscle cells. to determine the cellular mode of action of a changing pH and PCO2 on cerebral arterial muscle we will examine the effect of changing pH and PCO2 on isolated pressurized cerebral arteries. It is essential to delineate the mechanisms which control normal cerebral blood flow before we can fully understand the etiology of cerebral vascular pathologies such as stroke and impaired cerebral blood flow during ischemia.